Human protooncogene kg-20 and protein encoded therein

ABSTRACT

A human protooncogene having a base sequence of SEQ ID: 1 or a fragment thereof is overexpressed in various cancer tissues and can be used in diagnosing various cancers and an antisense gene complementary thereto can be used in treating cancers.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel protooncogene andprotein encoded therein, which can be used in diagnosis of variouscancers, preparation of transgenic animal, antisense gene therapy andanticancer drug development.

BACKGROUND OF THE INVENTION

[0002] Higher animals including man each carry approximately 100,000genes, but only about 15% thereof is expressed, and characteristics ofindividual's biological processes, e.g., genesis, differentiation,homeostasis, responses to stimuli, control of cell division cycle, agingand apoptosis (programmed cell death), are determined depending on whichgenes are expressed (Liang, P. and A. B. Pardee, Science 257: 967-971,1992).

[0003] Pathogenic phenomena such as tumorigenesis are caused by genemutation which brings about changes in the mode of gene expression.Therefore, comparative studies of gene expressions in various cells havebeen conducted to provide bases for establishing viable approaches tothe understanding of diverse biological phenomena.

[0004] It has been reported that tumorigenesis is caused by variousgenetic changes such as the loss of chromosomal heterozygosity,activation of oncogenes and inactivation of tumor suppressor genes,e.g., p53 gene (Bishop, J. M., Cell 64: 235-248, 1991; and Hunter, T.,Cell 64: 249-270, 1991). Further, it has been reported that 10 to 30% ofhuman cancer arises from the activation of oncogene throughamplification of protooncogenes.

[0005] Therefore, the activation of protooncogenes plays an importantrole in the etiology of many tumors and there has existed a need toidentify protooncogenes.

[0006] The present inventors have endeavored to unravel the mechanisminvolved in the tumorigenesis of cervical cancer; and, have unexpectedlyfound that novel protooncogenes, human cervical cancer proto-oncogene 1(HCCR-1) (Korea Patent Application No. 2000-16757 (Mar. 31, 2000)) andHCCR-2 (Korea Patent Application No. 2000-71202 (Nov. 28, 2000)), arespecifically overexpressed in cancer cells. Protooncogene causesquantitative and qualitative changes of the corresponding proteinproduct expression via a conformational change (Weinberg, R. A., CancerResearch 49:3713-3721, 1989). The conformationally changed-cancerprotein destroys signal transduction pathways in the cell surface,cytoplasm or nucleus; while in the absence of tumor suppressor geneproduct, it stimulates cell proliferation together with other cancerproteins, disturbs cell cycle, and destroys cell death (apoptosis)mechanism (Todd, R., Anticancer Research 19:4729-4746, 2000).

[0007] The yeast two-hybrid method, developed in 1989 by Fields et al.(Fields, S. and Song, O., Nature 340:245-246, 1989) for examining suchprotein-protein interactions, has been widely applied to elucidate thecell death mechanism (Wallach, D. et al., Curr. Opin. Immunol.10:131-136, 1989) as well as other biological processes using variousgenetic tools (Pandey, A. and Mann, M., Nature 405:837-846, 2000).

[0008] In the course of screening for a novel binding protein toprotooncogene KG-19 using the yeast two-hybrid method, the presentinventors have discovered a novel human protooncogene, KG-20 whichexpresses a protein that specifically binds to protooncogene KG-19.Protooncogene KG-20 can be advantageously used in diagnosis, preventionand treatment of various cancers, e.g., leukemia, lymphoma, colon,breast, kidney, stomach, lung, ovary and uterine cancers.

SUMMARY OF THE INVENTION

[0009] Accordingly, an object of the present invention is to provide anovel protooncogene and a fragment thereof.

[0010] Other objects of the present invention are to provide:

[0011] a recombinant vector containing said protooncogene or a fragmentthereof and a microorganism transformed therewith;

[0012] a protein encoded in said protooncogene and a fragment thereof;

[0013] a kit for diagnosing cancer which comprising said protooncogeneor a fragment thereof;

[0014] a kit for diagnosing cancer which comprising said protein or afragment thereof;

[0015] an antisense gene having a base sequence complementary to that ofsaid protooncogene or a fragment thereof;

[0016] a process for treating or preventing cancer by using saidantisense gene; and

[0017] a process for screening anticancer drug candidates by using saidprotooncogene or a fragment thereof.

[0018] In accordance with one aspect of the present invention, there isprovided a novel protooncogene having the nucleotide sequence of SEQ IDNO: 1 or a fragment thereof.

[0019] In accordance with another aspect of the present invention, thereis provided a recombinant vector containing said protooncogene or afragment thereof and a microorganism transformed with said vector.

[0020] In accordance with still another aspect of the present invention,there is provided a protein having the amino acid sequence of SEQ ID NO:2 or a fragment thereof derived from said protooncogene or a fragmentthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and other objects and features of the present inventionwill become apparent from the following description of the invention,when taken in conjunction with the accompanying drawings whichrespectively show;

[0022]FIG. 1A: the results of northern blot analyses for protoongeneKG-20 expressed in human cancer cell lines (promyelocytic leukemia HL-60cell, HeLa cervical cancer cell, chronic myelogenous leukemia K-562cell, lymphoblastic leukemia MOLT-4 cell, Burkitt's lymphoma Raji cell,SW480 colon cancer cell, A549 lung cancer cell and G361 melanoma cell);

[0023]FIG. 1B: the results obtained after hybridizing the same sample ofFIG. 1A with β-actin;

[0024]FIG. 2A: the phase-contrast feature of monolayer-cultured wildtype human embryonic kidney 293 cells;

[0025]FIG. 2B: the phase-contrast feature of monolayer-culturedKG-20-transfected human embryonic kidney 293 cells;

[0026]FIG. 3: hematoxylin-eosin staining of monolayer-culturedKG-20-transfected human embryonic kidney 293 cells;

[0027]FIG. 4: tumorigenicity of KG-20 cells in nude mouse;

[0028]FIG. 5: sodium dodecyl sulfate (SDS)-PAGE results showing proteinexpression patterns before and after the IPTG induction inKG-20-transfected human embryonic kidney 293 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The novel protooncogene of the present invention, designatedKG-20, consists of 622 base pairs and has the DNA sequence of SEQ IDNO: 1. This KG-20 protooncogene specifically binds to humanprotooncogene KG-19 (hereinafter “KG-19”) described in Korea PatentLaid-open Publication No. 2002-41553.

[0030] In SEQ ID NO: 1, the open reading frame corresponding to baseNos. 74 to 613 is a full-length protein encoding region and thepredicted amino acid sequence derived therefrom is shown in SEQ ID NO:2, which consists of 179 amino acids (“KG-20 protein”). However, inconsideration of the degeneracies of codons and the preferred codons ina specific animal wherein the protooncogene of the present invention isto be expressed, various changes and modifications of the DNA sequenceof SEQ ID NO: 1 may be made, e.g., in the coding area thereof withoutadversely altering the amino acid sequence of the expressed protein, orin the non-coding area without adversely affecting the expression of theprotooncogene. Therefore, the present invention also includes, in itsscope, a polynucleotide having substantially the same base sequence asthe inventive protooncogene, and a fragment thereof. As used herein,“substantially the same polynucleotide” refers to a polynucleotide whosebase sequence shows 80% or more, preferably 90% or more, most preferably95% or more homology to the protooncogene of the present invention.

[0031] The protein expressed from the protooncogene of the presentinvention consists of 179 amino acids and has the amino acid sequence ofSEQ ID NO: 2. The molecular weight of this protein is about 20 kDa. Theprotooncogene KG-20 of the present invention is different from the Homosapiens timing protein, CLK-1 (Vajo, Z. et al., Am. J. Hum. Genet. 61:A263-A263, 1997), in that the arginine residues at 34^(th) and 134^(th)position of CLK-1 are replaced with alanine and lycine, respectively, inSEQ ID NO: 2.

[0032] However, various substitution, addition and/or deletion of theamino acid residues of protein may be performed without adverselyaffecting the protein's function. Further, a portion of the protein maybe used when a specific purpose is to be fulfilled. These modified aminoacids and fragments thereof are also included in the scope of thepresent invention. Therefore, the present invention includes, in itsscope, a polypeptide having substantially the same amino acid sequenceas the protein derived from the oncogene of the present invention and afragment thereof. As used herein, “substantially the same polypeptide”refers to a polypeptide whose amino acid sequence shows 80% or more,preferably 90% or more, most preferably 95% or more homology to theamino acid sequence of SEQ ID NO: 2.

[0033] The protooncogene, or the protein, of the present invention canbe obtained from human placenta tissues or synthesized using aconventional DNA or peptide synthesis method. Further, the gene thusprepared may be inserted to a conventional vector to obtain anexpression vector, which may, in turn, be introduced into a suitablehost, e.g., a microorganism such as an E. coli or yeast, or an animalcell such as a mouse or human cell. A transformed cell withprotooncogene KG-20 is designated “KG-20 cell”.

[0034] A transformed host may then be used in producing the inventiveDNA or protein on a large scale. For example, E. coli DH5α istransformed with expression vector pCEV-LAC (Miki, T. et al., Gene 83:137-146, 1989) containing the inventive KG-20 gene to obtain an E. colitransformant designated DH5α/KG-20/pCEV-LAC which was deposited on Jun.2, 2001 with the Korean Collection for Type Cultures (KCTC) (Address:Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52,Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under theaccession number KCTC 1028BP, in accordance with the terms of BudapestTreaty on the International Recognition of the Deposit of Microorganismfor the Purpose of Patent Procedure.

[0035] In preparing a vector, expression-control sequences, e.g.,promoter, terminator, self-replication sequence and secretion signal,are suitably selected and combined depending on the host cell used.

[0036] The overexpression of the protooncogene of the present inventionoccurs in cancer tissues, e.g., leukemia, lymphoma, kidney, lung,stomach and skin cancer cell lines, suggesting that the inventiveprotooncogene induces such cancers. Further, when a normal humanembryonic kidney (HEK) 293 cell is transfected with the protooncogene ofthe present invention, an abnormal cell is produced. Morphologicalcharacterizations with optical and electronic microscopes show that theabnormal cell has the form of a tumor cell. By using thehematoxylin-eosin dye method, it can be confirmed that the tumor cell iscancerous (carcinoma).

[0037] When normal HEK 293 cells are transfected with the protooncogeneof the present invention and injected into the back of nude mice,tumorigenesis is observed after about 21 days from the injection, thetumor size becoming 2.0 cm×2.0 cm in 35 days.

[0038] Therefore, the protooncogene of the present invention is believedto be a factor common to all forms of cancers, and it can beadvantageously used in the diagnosis of various cancers and theproduction of a transgenic animal as well as in an antisense genetherapy and anticancer drug development.

[0039] A diagnostic method that can be performed using the protooncogeneof the present invention may comprise, for example, the steps ofhybridizing nucleic acids separated from the body fluid of a subjectwith a probe containing the protooncogene of the present invention or afragment thereof, and determining whether the subject has theprotooncogene by using a conventional detection method in the art. Thepresence of the protooncogene may be easily detected by labeling theprobe with a radioisotope or an enzyme. Therefore, a cancer diagnostickit containing the protooncogene of the present invention or a fragmentthereof is also included in the scope of the present invention.

[0040] A transgenic animal produced by introducing the protooncogene ofthe present invention into a mammal, e.g., a rat, is also included inthe scope of the present invention. In producing such a transgenicanimal, it is preferred to introduce the inventive protooncogene to afertilized egg of an animal before the 8th cell cycle stage. Thetransgenic animal can be advantageously used in screening forcarcinogens or anticancer agents such as antioxidants.

[0041] The present invention also provides an antisense gene which isuseful in a gene therapy. As used herein, the term “an antisense gene”means a polynucleotide comprising a base sequence which is fully orpartially complementary to the sequence of the mRNA which is transcribedfrom the protooncogene having the base sequence of SEQ ID NO: 1 or afragment thereof, said nucleotide being capable of preventing theexpression of the open reading frame (ORF) of the protooncogene by wayof attaching itself to the protein-binding site of mRNA.

[0042] The present invention also includes within its scope a processfor treating or preventing cancer by way of administering atherapeutically effective amount of the inventive antisense gene to asubject in need thereof.

[0043] In the inventive antisense gene therapy, the antisense gene ofthe present invention is administered to a subject in a conventionalmanner to prevent the expression of the protooncogene. For example, theantisense oligodeoxynucleotide (ODN) is mixed with a hydrophobizedpoly-L-lysine derivative by electrostatic interaction in accordance withthe method disclosed by Kim, J. S. et al. (J. Controlled Release 53:175-182, 1998) and the resulting mixed antisense ODN is administeredintravenously to a subject.

[0044] The present invention also includes within its scope ananti-cancer composition comprising the antisense gene of the presentinvention as an active ingredient, in association with pharmaceuticallyacceptable carriers, excipients or other additives, if necessary. Thepharmaceutical composition of the present invention is preferablyformulated for administration by injection.

[0045] The amount of the antisense gene actually administered should bedetermined in light of various relevant factors including the conditionto be treated, the chosen route of administration, the age and weight ofthe individual patient, and the severity of the patient's symptoms.

[0046] The protein expressed from the inventive protooncogene may beused in producing an antibody useful as a diagnostic tool. The antibodyof the present invention may be prepared in the form of a monoclonal orpolyclonal antibody in accordance with any of the methods well known inthe art by using a protein having the amino acid sequence of SEQ ID NO:2 or a fragment thereof. Cancer diagnosis may be carried out using anyof the methods known in the art, e.g., enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemicalstaining, western blot or immunoassay blot on polyacrylic gel, to asseswhether the protein is expressed in the body fluid of the subject.Therefore, a cancer diagnostic kit containing the protein having theamino acid sequence of SEQ ID NO: 2 or a fragment thereof is alsoincluded in the scope of the present invention.

[0047] A continuously viable cancer cell line may be established byusing the protooncogene of the present invention, and such a cell linemay be obtained, for example, from tumor tissues formed on the back of anude mouse by injecting fibroblast cells transformed with theprotooncogene of the present invention. The cell line thus prepared maybe advantageously used in searching for anti-cancer agents.

[0048] The present invention also includes within its scope a processfor screening anticancer drug candidates by using said protooncogene ora fragment thereof.

[0049] The following Examples and Test Examples are given for thepurpose of illustration only, and are not intended to limit the scope ofthe invention.

EXAMPLE 1 Yeast Two Hybrid Assay

[0050] To find a binding protein to human protooncogene KG-19 (GenebankAccession No: AF283671) protein product, yeast two hybrid assay wasperformed by using MATCHMAKER LexA Two-Hybrid System (ClontechLaboratories, Inc., U.S.A.) according to the previously reportedprocedure (Golemis, E. A., et al., Current Protocols in MolecularBiology, John Wiley & Sons, Inc. Chapters 20.0 and 20.1, 1996).

[0051] Cell lines and vectors used in the following experiment arelisted in Catalog No. K1609-1 Kit of Clontech Laboratories, Inc.

[0052] Yeast strain EGY48 was transformed with vector p8op-lacZ andcultured in an SD/-uracil/glucose plate, a synthetic dropout mediumhaving no uracil. The colony that grew in this plate was selected,cultured in an SD/-uracil/glucose medium, and transformed with a vectorprepared by inserting KG-19 gene into the SalI and BamHI sites of vectorpLexA as a ‘bait plasmid’.

[0053] To examine the expression of KG-19 gene cloned in vector pLexA,western blotting using LexA antibody was performed. The result showed a37 kDa band.

[0054] The colony expressing KG-19 gene was cultured in an SD/-uracil,histidine/glucose medium and transformed with vector pBD42AD which ishuman fetal brain-derived AD (activation domain) fusion library. Toconfirm the binding of the bait with the library, colony lifting assay(Breeden, L. and Nasmyth, K., Cold Spring Harbor Symposium Quant. Bio.50: 643-650, 1985) was performed. The interaction of the bait with thelibrary induced the formation of a blue colony in an X-gal plate. DNAwas purified from the yeast cultured using glass beads, and E. coli KC8was transformed with the purified DNA by electroporation and spreadingon an M9 minimal medium to select transformants. Plasmid DNA waspurified from the selected transformants and transformed into E. coliDH5α. Then, DNA purified from the selected E. coli transformants wassubject to HindIII digestion, to obtain a clone having 0.6 kb DNA.

EXAMPLE 2 Total cDNA Sequencing Analysis of Protooncogene KG-20

[0055] The clone obtained in Example 1 was subjected to sequencinganalysis using Sequenase version 2.0 DNA Sequencing Kit (United StatesBiochemical, Cleveland, Ohio, U.S.A.) according to the dideoxy chaintermination method. This clone was identified as a novel protooncogeneconsisting of 622 base pairs as described in SEQ ID NO: 1, and it wasdesignated “KG-20”. The nucleotide sequence of full-length KG-20 cDNAclone was registered at GenBank under Accession No. AF374413.

[0056] In SEQ ID NO: 1, the full open reading frame of KG-20corresponding to base Nos. 74 to 613 is a protein encoding region andthe predicted amino acid sequence derived therefrom is shown in SEQ IDNO: 2 which consists of 179 amino acids.

[0057] A λ pCEV containing the KG-20 gene was isolated from the phage λand cut by NotI to obtain an ampicillin-resistant pCEV-LAC. pCEV-LAC wasligated by DNA ligase to obtain a recombinant vector expressing KG-20gene. E. coli DH5α was transformed with the recombinant vector to obtaina transformed E. coli designated DH5α/KG-20/pCEV-LAC, which wasdeposited with Korean Collection for Type Cultures (Address: #52,Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea) on Jun. 2, 2001under the accession number of KCTC 1028BP.

EXAMPLE 3 Northern Blot Analysis

[0058] To determine the expression level of KG-20 gene in varioustissues, northern blot analyses were also carried out as recommended bythe supplier (Clontech) using a human cancer cell line sample containingpurified RNAs of 8 kinds of cancer cells blotted on a nylon membrane(FIGS. 1A and 1B).

[0059] The blots were hybridized overnight at 68° C. with ³²P-labeledrandom-primed KG-20 cDNA probe which was prepared using a Rediprime IIrandom prime labeling system (Amersham, England). The northern blotanalysis was repeated twice, and the results were quantified bydensitometry. The same blots were hybridized with a β-actin probe tostandardize the mRNA amount.

[0060]FIG. 1A shows the northern blot analysis for KG-20 gene expressedin human cancer cell lines, i.e., promyelocytic leukemia HL-60 cell,HeLa cervical cancer cell, chronic myelogenous leukemia K-562 cell,lymphoblastic leukemia MOLT-4 cell, Burkitt's lymphoma Raji cell, SW480colon cancer cell, A549 lung cancer cell, and G361 melanoma cell, usingKG-20 cDNA probe; and FIG. 1B, the same blot hybridized with a β-actinprobe. As can be seen in FIG. 1A, the 0.6 kb transcript of KG-20 isdetected at the Burkitt's lymphoma Raji, promyelocytic leukaemia HL-60,HeLa cervical cancer cell, chronic myelogenous leukaemia K-562,lymphoblastic leukaemia MOLT-4, SW480 colon cancer cell, A549 lungcancer cell, and G361 melanoma cell. Raji, in particular, shows a highertranscription level as compared with other cancer cells.

EXAMPLE 4 Construction of Expression Vectors and Transfection of AnimalCells

[0061] (Step 1) Preparation of a Vector Containing KG-20

[0062] An expression vector containing the coding region of KG-20 wasconstructed as follows.

[0063] First, fusion vector N-terminal pFlag-CMV constructed byinserting Flag gene into an eucaryotic expression vector pcDNA3(Invitrogen, U.S.A.) was digested with SalI/BamHI. KG-20 gene having thetotal coding sequence (corresponding to base Nos. 74-613 in SEQ IDNO: 1) was digested with SalI/BamHI, and the resulting SalI/BamHIfragment was inserted into the SalI/BamHI-digested N-terminal pFlag-CMV.Lipofectamine (Gibco BRL) was used to introduce the resulting vectorN-terminal pFlag-CMV/KG-20 to HEK 293 epithelial cell (ACTC CRL 1753,U.S.A.), followed by selection in a medium supplemented with G418(Gibco, U.S.A.). The resulting HEK 293 cell transfected with KG-20 wasdesignated “KG-20 cell”.

[0064] (Step 2) Human Embryonic Kidney 293 Epithelial Cell Transfectedwith Protooncogene KG-20

[0065] The wild type HEK 293 cells and KG-20 cells obtained in Step 1were each cultured in a monolayer in DMEM media containing 10% serum,and the growth features were observed with a Phase-contrast microscopy(Olympus, U.S.A.). FIGS. 2A and 2B show phase-contrast features ofmonolayer-cultured wild type HEK 293 cells and KG-20-transfected HEK 293cells. As shown in FIG. 2A, the wild-type HEK 293 cells proliferated asepithelial cells, each having a spindle-shape with a long, fine nucleusand meager cytoplasm. In case of KG-20 cells, the cell shape changesinto a polygonal form with an ovoid nucleus and plump cytoplasm, asshown in FIG. 2B.

[0066] Monolayer-cultured KG-20 cells stained with hematoxylin-eosin (H& E) exhibit nuclear pleiomorphism, distinct nucleoli, granularchromatin patterns, tumor giant cells and a typical mitotic figures asshown in FIG. 3.

EXAMPLE 5 Tumorigenicity of Protooncogene KG-20 in Animal

[0067] To analyze tumorigenicity, 5×10⁶ KG-20 cells were injectedsubcutaneously into the back of 9 nude mice (5-week-old athymic nu/nu onBALB/c background). All 9 mice injected with KG-20 cells showed palpabletumors after 21 days, the tumor size becoming 2.0 cm×2.0 cm in 35 daysas shown in FIG. 4.

EXAMPLE 6 Determination of Size of Protein Expressed After theTransfection of E. coli with Protooncogene KG-20

[0068] The full-length coding sequence region of protooncogene KG-20corresponding to base Nos. 74 to 613 of SEQ ID NO: 1, which encodesamino acid Nos. 1 to 179 of SEQ ID NO: 2, was inserted into BamHI/NotIrecognition site of the multiple cloning site of vector pGEX 4T-3(Amersham Pharmacia Biotech., U.S.A.) fused with GST gene, and theresulting vector pGEX 4T-3/KG-20 was transfected into E. coli BL21 (ATCC47092). The transfected E. coli was inoculated into an LB broth mediumand cultured in a rotary shaking incubator at 37° C. for 16 hours. Theculture solution was diluted with the same medium by 1/100(v/v) andincubated for 3 hours. 1 mM isopropyl β-D-thiogalacto-pyranoside (IPTG,Sigma) was added thereto to induce the protein synthesis.

[0069] The E. coli cells in the culture were disrupted by sonication andsubjected to gel electrophoresis using 12% sodium dodecyl sulfate (SDS)before and after the IPTG induction. FIG. 5 shows the SDS-PAGE resultwhich exhibits a protein expression pattern of the E. coli BL21 straintransfected with vector pGEX 4T-3/KG-20. After the IPTG induction, asignificant protein band was observed at about 46 kDa. This 46 kDafusion protein contained an about 26 kDa GST protein which was expressedfrom the gene of vector pGEX 4T-3.

[0070] While the embodiments of the subject invention have beendescribed and illustrated, it is obvious that various changes andmodifications can be made therein without departing from the spirit ofthe present invention which should be limited only by the scope of theappended claims.

1 2 1 622 DNA Homo sapiens CDS (74)..(610) 1 ggcgcgggaa ttcgattggatccggacaca ctttataatc tttccagttg atattgtcta 60 aagtcatgtc gac atg acttta gac aat atc aac ctg gca gct gtg 106 Met Thr Leu Asp Asn Ile Asn LeuAla Ala Val 1 5 10 gat cga ata atc cgg gtg gat cat gca ggc gaa tat ggagca aac cgc 154 Asp Arg Ile Ile Arg Val Asp His Ala Gly Glu Tyr Gly AlaAsn Arg 15 20 25 atc tat gcc ggg cag atg gct gtc ctg ggt cgg acc agc gtcggg cca 202 Ile Tyr Ala Gly Gln Met Ala Val Leu Gly Arg Thr Ser Val GlyPro 30 35 40 gtc att cag aaa atg tgg gat caa gaa aag gac cat ttg aaa aagttc 250 Val Ile Gln Lys Met Trp Asp Gln Glu Lys Asp His Leu Lys Lys Phe45 50 55 aat gag ttg atg gtt atg ttc agg gtc cgg cca aca gtt ctg atg ccc298 Asn Glu Leu Met Val Met Phe Arg Val Arg Pro Thr Val Leu Met Pro 6065 70 75 ttg tgg aac gtg ctg ggg ttt gca ctg ggg gcg ggg acc gcc ttg ctc346 Leu Trp Asn Val Leu Gly Phe Ala Leu Gly Ala Gly Thr Ala Leu Leu 8085 90 ggg aag gaa ggt gcc atg gcc tgc acc gtg gcg gtg gaa gag agc ata394 Gly Lys Glu Gly Ala Met Ala Cys Thr Val Ala Val Glu Glu Ser Ile 95100 105 gca cat cac tac aac aac cag atc agg acg ctg atg gag gag gac cct442 Ala His His Tyr Asn Asn Gln Ile Arg Thr Leu Met Glu Glu Asp Pro 110115 120 gaa aaa tac gag gaa ctt ctt cag ctg ata aag aaa ttt cgg gat gaa490 Glu Lys Tyr Glu Glu Leu Leu Gln Leu Ile Lys Lys Phe Arg Asp Glu 125130 135 gag ctt gag cac cat gac ata ggc ctc gac cat gat gca gaa ttg gct538 Glu Leu Glu His His Asp Ile Gly Leu Asp His Asp Ala Glu Leu Ala 140145 150 155 cca gcc tat gcc gtc ctg aag agc att atc cag gcc gga tgc agagtg 586 Pro Ala Tyr Ala Val Leu Lys Ser Ile Ile Gln Ala Gly Cys Arg Val160 165 170 gcg ata tat tta tca gaa aga tta taaagtgtgt cc 622 Ala IleTyr Leu Ser Glu Arg Leu 175 2 179 PRT Homo sapiens 2 Met Thr Leu Asp AsnIle Asn Leu Ala Ala Val Asp Arg Ile Ile Arg 1 5 10 15 Val Asp His AlaGly Glu Tyr Gly Ala Asn Arg Ile Tyr Ala Gly Gln 20 25 30 Met Ala Val LeuGly Arg Thr Ser Val Gly Pro Val Ile Gln Lys Met 35 40 45 Trp Asp Gln GluLys Asp His Leu Lys Lys Phe Asn Glu Leu Met Val 50 55 60 Met Phe Arg ValArg Pro Thr Val Leu Met Pro Leu Trp Asn Val Leu 65 70 75 80 Gly Phe AlaLeu Gly Ala Gly Thr Ala Leu Leu Gly Lys Glu Gly Ala 85 90 95 Met Ala CysThr Val Ala Val Glu Glu Ser Ile Ala His His Tyr Asn 100 105 110 Asn GlnIle Arg Thr Leu Met Glu Glu Asp Pro Glu Lys Tyr Glu Glu 115 120 125 LeuLeu Gln Leu Ile Lys Lys Phe Arg Asp Glu Glu Leu Glu His His 130 135 140Asp Ile Gly Leu Asp His Asp Ala Glu Leu Ala Pro Ala Tyr Ala Val 145 150155 160 Leu Lys Ser Ile Ile Gln Ala Gly Cys Arg Val Ala Ile Tyr Leu Ser165 170 175 Glu Arg Leu

What is claimed is:
 1. A human KG-20 protooncogene having the basesequence of SEQ ID NO: 1 or a fragment thereof.
 2. The protooncogene orfragment of claim 1, wherein the fragment has a base sequencecorresponding to base Nos. 74 to 613 of SEQ ID NO:
 1. 3. A proteinhaving the amino acid sequence of SEQ ID: 2 or a fragment thereof.
 4. Avector comprising the protooncogene or fragment of claim
 1. 5. Amicroorganism transformed with the vector of claim
 4. 6. Themicroorganism of claim 5, which is E. coli DH5α/KG-20/pCEV-LAC(Accession No.: KCTC 1028BP).
 7. A process for preparing the protein orfragment of claim 3 comprising culturing the microorganism of claim 5 or6.
 8. A kit for diagnosing cancer which comprises the protooncogene orfragment of claim 1 or
 2. 9. A kit for diagnosing cancer which comprisesthe protein or fragment of claim
 3. 10. An antisense gene having a basesequence which is complementary to the sequence of the full or partialmRNA transcribed from the protooncogene or fragment of claim 1 or 2 andbeing capable of binding the mRNA to inhibit the expression of saidprotooncogene or fragment.
 11. A composition for preventing or treatingcancer which comprises a therapeutically effective amount of theantisense gene of claim 10 and a pharmaceutically acceptable carrier.